Borrelia burgdorferi is the causative agent of Lyme disease, a global tick-borne infectious disease with an estimated 300,000 cases each year in the U.S. alone. Surface lipoproteins are playing a major role in maintenance of the natural tick-host enzootic cycle as well as in the pathogenesis of human Lyme disease. This proposal will investigate the mechanisms that B. burgdorferi uses to deploy its surface lipoprotein virulence factors.

The overall goal of the planned proposal will be to better understand the secretion mechanisms of major bacterial envelope proteins. Our model organism, the Lyme disease spirochete Borrelia burgdorferi, expresses an extraordinary number of outer membrane lipoproteins with various crucial functions, but their secretion pathways remain largely undefined. The product of B. burgdorferi open reading frame BB0346 can be modeled on LolA (Lipoprotein outer membrane localization A) chaperones of gram-negative bacteria, but BB0346 shares only about 20% amino acid identity with other members of the LolA protein superfamily. Thus, some structural and functional differences are likely.

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In Aim 1 of this one-year pilot project, we plan to express, purify, crystallize and determine the structure of the B. burgdorferi LolA homolog BB0346. Recombinant expression systems and purification approaches are currently being optimized. Structure determination should be possible by X-ray crystallography and molecular replacement using the two known structures of gram-negative LolA proteins. The obtained structural data will lay the groundwork for the characterization of lipoprotein-BB0346 complexes.

In Aim 2, we plan to initiate a screen for inhibitors of lipoprotein secretion in B. burgdorferi by adapting an already established 96-well-plate protein localization screen using fluorescent B. burgdorferi lipoprotein fusions. Any initial lead compounds will be analyzed for their spatiotemporal action affecting lipoprotein secretion using established B. burgdorferi protein localization assays.

Overall, the proposal will combine two independent but synergistic approaches to yield novel information that will significantly impact our understanding of bacterial protein secretion in a medically important model organism. The experimental plan will take advantage of our own longstanding expertise with B. burgdorferi lipoproteins and the excellent environment provided by the Protein Structure and High Throughput Screening cores at KU Lawrence.